Project Summary/Abstract ? Project 2 Immunotherapy with immune checkpoint inhibitors (ICI) is revolutionizing the treatment of many cancers, including non-small cell lung cancer (NSCLC) where a small subset of patients with metastatic disease have significant responses. The antitumor activity of ICI is thought in part to be mediated by CD4+ and CD8+ T cells that recognize neoantigens, which are peptides derived from mutations in expressed genes in tumor cells and presented by class I or II MHC molecules. Thus, the failure of most patients to respond to ICI may result from an insufficient pre-existing tumor-specific T cell response, irreversible dysfunction of previously activated T cells, or local immunosuppressive mechanisms. A therapeutic vaccine capable of boosting or inducing de novo functional T cell responses to neoantigens could be beneficial alone, or in combination with ICI or other modalities that overcome immunosuppression in the tumor microenvironment. Putative neoantigens are prevalent in NSCLC due to the high mutation burden, and may be superior to self-antigens as vaccine targets because the T cell repertoire capable of responding is not affected by central tolerance mechanisms. Moreover, multiple neoantigens can theoretically be targeted by a vaccine, which could overcome heterogeneity in antigen and MHC expression on tumors, and in the quality of a single neoantigen. Multiple candidate neoantigens can be identified using whole exome sequencing of tumors to detect coding mutations, and algorithms that predict peptides likely to bind to MHC molecules. Initial clinical applications of therapeutic neoantigen vaccines in melanoma have recently provided proof-of-principle, and revealed the potential of this personalized approach to cancer immunotherapy. We have developed a novel approach to neoantigen vaccination that utilizes the systemic administration of autologous T cells engineered to express cancer-specific mutations (Tvax). This strategy was suggested by clinical data from our lab showing that adoptive transfer of human T cells expressing transgenes encoding foreign proteins induced potent CD8+ and CD4+ T cell responses specific for the transgene product that were boosted by subsequent infusions, even in patients with severely compromised immunity. T cells provide a versatile platform for personalized medicines, including cell based vaccines because they can be easily genetically modified and expanded in cGMP conditions, safely administered systemically, and traffic efficiently to lymph node sites to deliver antigens where immune responses are initiated. This project will translate this unique approach for vaccination to neoantigens in preclinical models and patients with NSCLC.